Stem cells (pluripotent stem cells) having the ability to differentiate into cells of multiple lineages are known to be obtained from various tissues. Mesenchymal stem cells isolated from bone marrow are one of such groups of cells and have the ability to differentiate into various cells such as osteocytes, cardiomyocytes, chondrocytes, and adipocytes (Patent Documents 1 to 4). Using this differentiation ability, attempts have been made to apply mesenchymal stem cells in regenerative treatment of various tissues. For example, it has been reported that administration of mesenchymal stem cells to patients with myocardial infarction was tested aiming to regenerate the cardiac muscle cells which had undergone necrosis in myocardial infarction, resulting in an improvement of the patients' cardiac function (Non-patent Documents 1).
It has also been known that mesenchymal stem cells can suppress T-cell mediated immune responses when administered to the living body, and thus can be used as a pharmaceutical agent to suppress rejection reaction after transplantation (Patent Document 5). Focusing on the mesenchymal stem cells' suppressive effect on rejection reaction, clinical studies are being conducted to investigate the clinical efficacy of human mesenchymal stem cells on graft-versus-host disease (GVHD) occurring after bone marrow transplantation (Non-patent Documents 2 and 3). In those clinical studies, human mesenchymal stem cells allogeneic to the patients have been used.
Use of mesenchymal stem cells has also been advocated for angiogenesis, autoimmune diseases, inflammatory responses (in Alzheimer's disease, Parkinson's disease, stroke, brain cells damage, psoriasis, chronic dermatitis, contact dermatitis, arthritis including osteoarthritis, rheumatoid arthritis, and the like, inflammatory bowel disease, chronic hepatitis), cancer, allergic diseases, sepsis, trauma (burn, surgery, transplantation), inflammation of tissues and organs (cornea, lens, pigment epithelium, retina, brain, spinal cord, uterus during pregnancy, ovaries, testes, adrenal gland) (Patent Document 6). However, in this literature, the efficacy of the mesenchymal stem cells has been examined in vitro only, and in vivo efficacy of the mesenchymal stem cells has not been evaluated.
Mesenchymal stem cells isolated from bone marrow have high proliferation potency in artificial media, and like other cultured cells, they can be stored and supplied in frozen state (Patent Document 7). Therefore, in clinical trials of allogeneic human mesenchymal stem cells against GVHD, mesenchymal stem cells are supplied to medical institutions in a frozen state, and are thawed and used immediately before administration to the patients.
Mesenchymal stem cells (MSCs) are known to be obtained not only from bone marrow but also from various tissues such as adipose tissue (Patent Document 8), umbilical cord and placental tissues (Patent Document 9). Mesenchymal stem cells (or other similar cells) can also be obtained from various dental tissues such as dental pulp, dental follicle (Patent Document 14), dental sac (Patent Documents 15 and 20), dental papilla (Patent Document 16), periodontal ligament (Patent Document 17). These mesenchymal stem cells are supposed to generally possess the ability to differentiate into adipocytes.
Mesenchymal stem cells from dental pulp are obtained roughly following this procedure (Patent Document 19). Namely, a tissue obtained through crushing an extracted tooth is treated with type I collagenase and dispase, and cell clumps are removed by filtration to obtain a cell suspension. The cells then are allowed to proliferate in a culture flask using a DMEM medium containing 20% FBS. The cells which proliferated and adhered to the inner surface of the culture flask are detached by trypsin treatment and recovered. The cells thus recovered are the mesenchymal stem cells. A serum-free medium for culturing mesenchymal stem cells derived from dental pulp has also been reported (Patent Document 18).
Dental pulp is a loose fibrous connective tissue that occupies the dental pulp cavity of tooth, and is divided into coronal pulp and radicular pulp depending on their location. In addition, some pluripotent undifferentiated cells except mesenchymal stem cells may possibly occur in the dental pulp. Therefore, pluripotent stem cells derived from other undifferentiated cells than mesenchymal stem cells and having different characteristics from mesenchymal stem cells may possibly be obtained from dental pulp. This possibility is supported by a report stating that stem cells derived from dental pulp do not differentiate into adipocytes (Patent Document 11). Namely, it is considered that stem cells obtained from dental pulp include at least two types which are distinguished from each other based on their ability or inability to differentiate into adipocytes. It has also been reported that stem cells obtained from the dental pulp of deciduous teeth have different properties from those occurring in the dental pulp of permanent teeth in that they, for example, have higher proliferative ability and exhibit higher expression levels of FGF2, TGF-β, collagen I and collagen III compared with those obtained from dental pulp of permanent teeth (Patent Document 19). Further there is a report showing that pluripotent stem cells derived from dental pulp differ from bone marrow-derived mesenchymal stem cells in their property when induced to differentiate into osteoblasts (Patent Document 20).